Fluorescence imaging using a fluorescent protein is an extremely effective method in the field of life science. For example, morphologies of various tissues, localization of a protein, etc. can be easily observed with use of a transgenic animal in which a fluorescent protein is expressed with use of a suitable promoter. However, a tissue normally has a considerably large thickness. Thus, it has been conventionally necessary to prepare a physical section so as to observe localization of fluorescence at a depth of the tissue.
Confocal microscopes and multiphoton excitation microscopes each for use in deep fluorescence imaging have been widespread in recent years. For example, use of multiphoton excitation microscopes allows deep imaging of approximately 300 μm to 700 μm, but more than 700 μm in depth is difficult to observe.
Several tissue clearing methods have been developed so far so that a deeper part can be observed. Known examples of the tissue clearing methods encompass a method using an aromatic organic solvent such as BABB (mixture of benzyl alcohol and benzyl benzoate 1:2 ratio), methyl salicylate, or dibenzyl ether (Non Patent Literature 1, Non Patent Literature 2, Non Patent Literature 3, and Non Patent Literature 4).
A group of Kleinfeld et al. showed in 2009 that up to 1.5 mm in depth can be observed with use of a multiphoton microscope by immersing a brain tissue in a 75% (w/v) sucrose aqueous solution (Non Patent Literature 5). A tissue clearing reagent named FocusClear™ (product name) is also commercially available (Patent Literature 1).
Further, a clearing reagent as which an aqueous solution containing urea and glycerin is employed has recently been developed (Non Patent Literature 6). This clearing reagent which is combined with a multiphoton microscope allows imaging of up to 4 mm in depth for a brain tissue.